Anthrax is a zoonotic disease whose etiologic agent is a gram-positive sporulating bacterium, B. anthracis. Human beings can acquire it via infected animals or contaminated animal products. The etiologic agent of anthrax (Bacillus anthracis) is a potential threat as an agent of biowarfare or bioterrorism because exposure to aerosolized B. anthracis spores can be lethal to mammals, such as humans. Vaccination is currently thought of as the most effective way to protect individuals and entire populations from an anthrax infection. Virulence of B. anthracis is due to two major antigens: the antiphagocytic capsular antigen and the anthrax toxin. The antiphagocytic capsular antigen does not protect against anthrax infection. However, the anthrax toxin is highly immunogenic and is the basis for successful anthrax vaccines.
The anthrax toxin has three peptide components: the protective antigen (PA; 83 kDa), the lethal factor (LF; 90 kDa), and the edema factor (EF; 89 kDa). The EF is a calcium-calmodulin-dependent adenylate cyclase believed to cause the edema associated with anthrax infection and to prevent immune cells from ingesting and degrading the bacteria. The LF is a cell-type specific metalloprotease that cleaves mitogen-activated protein kinase-kinases and several peptide hormones. It causes macrophage cell death and release of toxic substances (e.g., those associated with septic shock such as TNF-α and IL-1). LF is the major virulence factor associated with anthrax toxicity and is responsible for systemic shock and death. The genes for all three peptide components have been cloned and sequenced. No single anthrax toxin component alone is toxic; however, a combination of PA and either LF or EF leads to infection and pathogenesis. During the B. anthracis infectious process, PA83 binds to a ubiquitous cell surface receptor. One or more proteases including a furin-like protease is present at the exterior of cells and plays a role in the proteolytic activation of receptor bound PA. PA is secreted as an 83 kDa monomeric polypeptide. Monomeric PA binds to a mammalian cell surface receptor and is proteolytically cleaved. The C-terminal 63 kDa fragment (PA63) remains bound to the cell and the N-terminal 20 kDa (PA20) dissociates from PA63. The cleavage generates PA63 and exposes a high affinity site on PA to which LF/EF can bind competitively. PA63 heptamerizes and inserts into the membrane as a pore upon exposure to acidic pH after receptor mediated endocytosis. The PA63 oligomer translocates EF/LF into the cytosol. The fourth domain of PA (PA-D4) is responsible for initial binding of the anthrax toxin to the cellular receptor, and is an attractive target for vaccines.
Some studies have illustrated that both monoclonal and polyclonal antibodies to PA may neutralize the anthrax toxin and function to provide immunity against the pathogen. One such current anthrax vaccine includes an aluminum hydroxide-adsorbed cell-free filtrate of cultures of a noncapsulating nonproteolytic strain of B. anthracis (Anthrax Vaccine Absorbed, AVA) in which PA is the major protective component.
Although these vaccines have proven efficacious, they possess certain limitations. Namely, vaccine quality and efficacy vary among production batches depending on the levels of PA production and the presence of impurities, such as traces of active toxin components LF and EF, which can produce serious side effects in a subject.
Culture supernatants of B. anthracis have been the major source for purifying PA. However, working with B. anthracis cultures requires expensive biosafety level-3 containment facilities. Additionally, PA preparation from B. anthracis is often contaminated with LF or EF. Heterologous expression of PA from other hosts, such as Bacillus subtilis, has been attempted in the past, but with difficulty. PA production from B. subtilis or a protease deficient B. subtilis host yields only limited quantities of PA, thus increasing the costs of additional production batches. Similarly, Baculovirus vectors have also been used to express PA in insect cells; however, purification is not feasible due to low PA yields and the persistence of undesirable impurities. PA has also been expressed in Escherichia coli, however, a low yield was observed and the protein was insoluble when expressed in the cytoplasm. Heterologous expression in E. coli used codon optimized recombinant PA (rPA) and the protein was targeted into the periplasm of the expression host.